The present invention relates to processes for the extraction and purification on an industrial scale of commercially useful enzymes which can be derived from the bacterium Zymomonas mobilis (hereinafter for convenience referred to simply as Z. mobilis), in containing high levels of enzymes of the Entner-Doudoroff pathway and being an excellent source for isolating these enzymes, besides having associated enzymes at high levels which are involved in the fermentation of sugars to alcohol.
Enzymes which can be extracted from Z. mobilis and purified on an industrial scale by the processes of the invention are as follows:
Glucose 6-phosphate dehydrogenase EC 1.1.1.49 PA1 Glucokinase EC 2.7.1.2 PA1 Fructokinase EC 2.7.1.4
Pyruvate kinase EC 2.7.1.40
each of which has an established commercial usefulness.
Glucose 6-phosphate dehydrogenase, which is currently available from biochemical reagent suppliers, is useful as a component in clinical diagnostic reagents for determining glucose, ATP, other sugars, and for enzyme activity measurements. These uses extend to the industrial field, specifically the food industry, with respect to analytical methods for estimating sugars, and to the biochemical field, with respect to forensic science, genetics and microbiology research for enzyme activity measurements, measurements of sugars, ATP, and the like.
Glucokinase, which is likewise available, is useful specifically for clinical and food analysis for measuring glucose, ATP, and certain enzymes such as creatine kinase in clinical diagnostic tests. Pyruvate kinase, which is likewise available, is useful specifically for enzyme activity measurements when the enzyme concerned is a kinase, its use mainly being in clinical and biological research, however, it is also valuable in an ATP-regeneration system for chemical and biochemical synthesis where ATP is consumed in the reaction. Fructokinase, which is likewise available, is useful specifically for measuring fructose in the presence of glucose, which is a test valuable in the food industry.
Glucose 6-phosphate dehydrogenase is present in microbial cells other than Z. mobilis, for instance, Leuconostoc mesenteroides (hereinafter referred to simply as L. mesenteriodes for convenience) or other microorganisms containing NAD-specific glucose 6-phosphate dehydrogenase. Extraction and purification of the enzyme on an industrial scale from such cells presents problems and involves many technical difficulties.
Industrial production of intracellular enzymes can be considered to involve three distinct phases, namely: (i) propagation of the microbial cells and their recovery in paste form from the propagation medium; (ii) extraction of the microbial cells paste to provide the raw material for isolation of the enzymes; and (iii) isolation and purification of the enzymes from the extract of that raw material.
Methods for the propagation of microbial cells such as L. mesenteroides and Z. mobilis are known. Also, several different methods have been described for the extraction of microbial cells. For instance, physicochemical methods involve osmotic shock [Schwinghamer, E. A. (1980) FEMS Microbiology Letters. 7, 157-162], lysozyme treatment [Saint-Blacard, J., Mawal, J. P. Constant, J. F., Berthon, J. and Jolles, P. (1981) Bioscience Reports. 1, 119-123], organic solvents [Rutter, W. J. and Hunsley, J. R. (1966) Methods Enzymol. 9, 480-486 ], and detergents [Miozari, G. F., Niederberger, P. and Hutter, R. (1978) Analyt. Biochem. 90, 220-233]. Mechanical methods can also be used successfully.
The ability of microbial cells such as L. mesenteroides and Z. mobilis cells to lyse and release enzymes into solution is largely dependent upon the conditions under which those cells are grown. For example, when those cells are harvested in conditions of (i) high temperature, (ii) high alcohol level, and (iii) low pH, they are more likely to lyse easily than if the conditions are more moderate.
In that alcohol is the product of Z. mobilis fermentation, high yields of cells can only be obtained in the presence of alcohol, however, the temperature and pH of the cultivation media may be varied according to the degree of strength required in the cells to resist lysis prior to harvesting.
Thus, a problem with purposely growing so-called "weaker" cells is that they are liable to lyse prior to harvesting and during transportation for extraction, thereby reducing yield of the desired enzyme. On the other hand, so-called "tough" cells are much more difficult to lyse during harvesting. A reliable method for extracting both "weak" and "tough" cells equally well is needed.
In that somewhat equivalent problems to those indicated above in relation to the production and extraction of Z. mobilis cells for recovery of the enzyme glucose 6-phosphate dehydrogenase, apply for the recovery of the other enzymes listed above, a reliable process for extracting those enzymes from said cells, also is needed.